Neurons communicate with one another by releasing neurotransmitters, peptides, and small molecules by exocytosis. The fusion of neurotransmitter-containing vesicles with the plasma membrane is driven by a core set of proteins known as SNAREs along with several regulatory factors. While the identity of many of these proteins and their requirement in calcium-triggered exocytosis has been demonstrated, the underlying molecular mechanism by which SNAREs and SNARE-associated proteins function is still unknown. A number of fundamental questions remain: How is the core SNARE fusion machinery activated to drive membrane fusion? What are the orientations, architectures, and stoichiometries of the proteins that constitute the fusion machine? What are the underlying structural rearrangements of the proteins associated with membrane fusion? To address these and related questions, we will employ a combination of novel fluorescence techniques, biochemistry, and live cell imaging. We will focus our efforts at two levels of investigation, studying the structure and dynamics of SNAREs in isolated membrane patches as well as in intact living cells. In this grant two specific aims are proposed: 1) map the orientation of the plasma membrane resident t-SNARE Syntaxin and Syntaxin binding proteins relative to each other and to the plasma membrane with patch-clamp fluorometry;and 2) characterize the stoichiometry, dynamic associations, and functional roles of SNAREs and SNARE-associated enzymes in vivo with total internal reflection fluorescence (TIRF) microscopy of single exocytic vesicles and fluorescently-tagged proteins. These studies will map the precise molecular steps required to activate SNAREs and drive calcium-triggered membrane fusion in neurons and endocrine cells. A detailed understanding of how these enzymes function at the atomic level will provide insights into the control and regulation of synaptic transmission, how its malfunction leads to disease, and how its modulation might be targeted for novel therapies.